Process, Vectors and Engineered Cell Lines for Enhanced Large-Scale Transfection

ABSTRACT

Processes, vectors and engineered cell lines for large-scale transfection and protein production in mammalian cells, especially Chinese Hamster Ovary (CHO) cells are described in which transfection efficiencies are realized through the use of a single vector system, the use of functional oriP sequences in all plasmids, the use of codon-optimized Epstein-Barr virus nuclear antigen-1 (EBNA1) constructs, the use of a fusion protein between a truncated Epstein-Barr virus nuclear antigen-1c (EBNA1c) protein and a herpes simplex virus protein VP16, the use of a 40 kDa fully deacetylated poly(ethylenimine) as a transfection reagent, the use of co-expression of a fibroblast growth factor (FGF) and/or the use of protein kinase B to potentiate heterologous gene expression enhancement by valproic acid (VPA).

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a division of U.S. patent application Ser. No.12/989,898 filed Oct. 27, 2010, currently pending, which is acontinuation-in-part of U.S. patent application Ser. No. 11/576,005filed Mar. 26, 2007, currently pending, and a national stage filingunder 35 U.S.C. §371 of international application PCT/CA2009/000263filed Mar. 9, 2009, which claims the benefit under 35 U.S.C. 119(e) ofU.S. provisional patent application Ser. No. 61/071,760 filed May 15,2008, the entire contents of all of which are herein incorporated byreference.

FIELD OF THE INVENTION

The present invention relates to processes, vectors and engineered celllines for large-scale transfection and protein production.

BACKGROUND OF THE INVENTION

Large-scale transfection of Chinese Hamster Ovary (CHO) cells withcost-effective reagents for the production of r-proteins suffers fromlow efficiency and low productivity. In addition, plasmid vectors usedin CHO cells are not fully optimized for transient gene expression.

There are some very efficient and commercially available cationic lipidsformulation that can be used to transfect CHO cells in serum-freemedium, for example FreestyleMax™ from Invitrogen. However, thesecationic lipids are very expensive. Also, to improve productivity, it isbecoming current practice to lower the cultivation temperature followingtransfection to prolong the production phase and to enhanceproductivity. This temperature shift is not “user friendly” when workingat large-scale or when using non-refrigerated culture devices (Wavebioreactors, etc). Also, the exact temperature at which the shifts aredone may be critical for getting optimal enhancement (e.g. 29 vs. 30 vs.31 vs. 32 degrees Celsius).

International patent publication WO 2007/048601 reports an expressionsystem in CHO cells stably expressing EBNA1 for the production ofr-proteins. However, this document specifically admonishes that the celllines shall not contain a functional copy of the Epstein-Barr virus(EBV) oriP sequence. Further, the full length EBNA1 structural geneencoding a full length EBNA1 protein is transfected into the cell line,and the oriP sequence is never in the same vector as the EBNA1 geneconstruct.

International patent publication WO 2002/090533 describes enhancedproduction of recombinant proteins by transient transfection ofsuspension-growing mammalian cells. However, only full length EBNA1structural genes are used encoding full length EBNA1 proteins and onlytransient expression of a gene of interest is achieved.

International patent publication 2006/096989 describes expressionvectors for enhanced transient gene expression and mammalian cellsexpressing them. However, only HEK293 cell lines are exemplified and theexpression system used does not contain both the EBNA1 gene constructand the oriP sequence in the same vector. Further, only transientexpression of a gene of interest is achieved.

There is a need in the art for processes, vectors and engineered celllines for more efficient and productive transfection of cells at a largescale.

SUMMARY OF THE INVENTION

In accordance with a first aspect of the present invention, there isprovided an expression system for stable expression of a gene ofinterest, the expression system comprising one vector having anEpstein-Barr virus nuclear antigen-1 (EBNA1) nucleotide sequenceencoding a truncated EBNA1 protein, a promoter and a polyadenylationsignal for the EBNA1 nucleotide sequence, an oriP nucleotide sequence,the gene of interest and a promoter and a polyadenylation signal for thegene of interest.

In accordance with a second aspect of the present invention, there isprovided a method of stably expressing a gene of interest in mammaliancells, the method comprising: transfecting a mammalian cell with avector having an Epstein-Barr virus nuclear antigen-1 (EBNA1) nucleotidesequence encoding a truncated EBNA1 protein, a promoter and apolyadenylation signal for the EBNA1 nucleotide sequence, an oriPnucleotide sequence, the gene of interest and a promoter and apolyadenylation signal for the gene of interest; and, replicating thecell to provide mammalian cells that stably express the gene ofinterest.

In accordance with a third aspect of the present invention, there isprovided an expression system for stable expression of a gene ofinterest, the expression system comprising: a first vector having anEpstein-Barr virus nuclear antigen-1 (EBNA1) nucleotide sequenceencoding a truncated EBNA1 protein, a promoter and a polyadenylationsignal for the EBNA1 nucleotide sequence and an oriP nucleotidesequence; and, a second vector having a gene of interest, a promoter anda polyadenylation signal for the gene of interest and an oriP nucleotidesequence.

In accordance with a fourth aspect of the present invention, there isprovided a method of stably expressing a gene of interest in mammaliancells, the method comprising transfecting a mammalian cell with: a firstvector having an Epstein-Barr virus nuclear antigen-1 (EBNA1) nucleotidesequence encoding a truncated EBNA1 protein, a promoter and apolyadenylation signal for the EBNA1 nucleotide sequence and an oriPnucleotide sequence; and, a second vector having the gene of interest, apromoter and a polyadenylation signal for the gene of interest and anoriP nucleotide sequence to provide mammalian cells that stably expressthe gene of interest.

In the fourth aspect, transfecting the cell with the first and secondvectors may be accomplished simultaneously, or the cell may betransfected by one of the vectors first to produce a stable clonefollowed by transfection with the other vector to produce a clone thatstably expresses the gene of interest.

In accordance with a fifth aspect of the present invention, there isprovided a method of transiently expressing a gene of interest inChinese Hamster Ovary (CHO) cells, the method comprising: transfecting aCHO cell with a first vector having an Epstein-Barr virus nuclearantigen-1 (EBNA1) nucleotide sequence encoding a truncated EBNA1protein, a promoter and a polyadenylation signal for the EBNA1nucleotide sequence and an oriP nucleotide sequence, and a second vectorhaving the gene of interest and a promoter and a polyadenylation signalfor the gene of interest; and, replicating the cell to provide CHO cellsthat transiently express the gene of interest.

In the fifth aspect, transfecting the CHO cell with the first and secondvectors may be accomplished simultaneously, or the CHO cell may betransfected by one of the vectors first to produce a clone followed bytransfection with the other vector to produce a clone that transientlyexpresses the gene of interest.

In accordance with a sixth aspect of the present invention, there isprovided a use of a codon-optimized Epstein-Barr virus nuclear antigen-1(EBNA1) nucleotide sequence in an expression system for expressing agene of interest in mammalian cells.

In accordance with a seventh aspect of the present invention, there isprovided a fusion protein comprising: a truncated Epstein-Barr virusnuclear antigen-1c (EBNA1c) protein; and, a herpes simplex virus proteinVP16.

In accordance with an eighth aspect of the present invention, there isprovided a use of a 40 kDa fully deacetylated poly(ethylenimine) as atransfection reagent for improving transfection efficiency intransfection of Chinese Hamster Ovary (CHO) cells.

In accordance with a ninth aspect of the present invention, there isprovided a use of co-expression of a fibroblast growth factor (FGF) toincrease heterologous gene expression in Chinese Hamster Ovary (CHO)cells.

In accordance with a tenth aspect of the present invention, there isprovided a use of protein kinase B to potentiate valproic acid (VPA) toincrease heterologous gene expression in mammalian cells.

Further features of the invention will be described or will becomeapparent in the course of the following detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

In order that the invention may be more clearly understood, embodimentsthereof will now be described in detail by way of example, withreference to the accompanying drawings, in which:

FIG. 1 depicts schematic diagrams of EBNA1constructs.

FIG. 2 depicts graphs of SEAP activity in CHO cells co-transfected withpTT vectors encoding various EBNA1constructs.

FIG. 3A depicts genetic maps of pTT vectors encoding EBNA1c in cis undera strong (EF1α-HTLV; pTT31) or a weak (RSV; pTT34) promoter.

FIG. 3B depicts a graph of SEAP activity in CHO cells transfected withpTT vectors encoding EBNA1c in cis under a weak or strong promoter.

FIG. 4A depicts a Western blot of extracts from CHO cells transfectedwith a linearized pTT-EBNA1c vector containing a blasticidin resistancecassette.

FIG. 4B depicts a Western blot of aliquots of cultures of the CHO cellsof FIG. 4A at various time intervals without selection pressure.

FIGS. 5A-C depict Western blots of codon optimized EBNA1 expression inHEK293 and CHO cells.

FIG. 5D depicts a graph of secreted alkaline phosphatase (SEAP) activityin CHO cells co-expressing VP16-EBNA1c fusion protein compared tocontrol cells or cells expression an EBNA1c protein.

FIG. 6 depicts graphs of SEAP activity in HEK293 and CHO cellstransfected with pTT plasmids using LPEI or LPEI-Max.

FIG. 7 depicts a graph of SEAP activity in transiently transfectedCHO-EBNA1c cells illustrating the transactivating activity of oriP.

FIG. 8 depicts a graph of SEAP activity in transfected CHO-EBNA1c cellsillustrating the effect of the CMV5 promoter vs. the Elongation Factor 1alpha-HTLV (EF1α-HTLV) hybrid promoter.

FIG. 9 depicts graphs of SEAP activity illustrating the effect of FGF2(bFGF) co-expression on transient gene expression in HEK293-EBNA1t andCHO-EBNA1c cells.

FIG. 10 depicts a graph of SEAP expression in CHO-EBNA1c cellsillustrating protein kinase B (AKT) potentiation of valproic acid (VPA)enhancement of transient gene expression.

FIG. 11 depicts genetic maps of SEAP-encoding pTT plasmids pTT22 (+oriP)and pTT30 (oriP) and graphs of SEAP activity in CHO-EBNA1c cellstransfected with linearized pTT22 and pTT30 plasmids illustrating theeffect of oriP on linearized vectors in CHO-EBNA1c cells.

FIG. 12 depicts a graph of SEAP activity in CHO-EBNA1c cells afterintegration of linearized pTT22 (+oriP) and pTT30 (-oriP) plasmids intheir chromosomes.

DESCRIPTION OF PREFERRED EMBODIMENTS Materials and Methods:

Cell culture: CHO cells are grown at 37° C. in FreestyleCHO medium(Invitrogen) supplemented with 8 mM glutamine in Erlenmeyer flasksshaken at 120 rpm in a humidified atmosphere containing 5% CO₂. Cellsare routinely maintained between 0.1×10⁶ and 3.0×10⁶ cells/ml.HEK293-EBNA1cells (clone 6E) are grown at 37° C. in F17 medium(Invitrogen) supplemented with 4 mM glutamine and 0.1% pluronic F68 inErlenmeyer flasks shaken at 120 rpm in a humidified atmospherecontaining 5% CO₂. Cells are routinely maintained between 0.1×10⁶ and2.0×10⁶ cells/ml.

Cell transfection: For transfection, CHO or HEK293 cells are grown inmaintenance medium until they reach a density of 1.5×10⁶ to 2.0×10⁶cells/ml and then the transfection mixture is added to the cells. Forevery ml of HEK293 cells to be transfected, 1 μg of plasmid DNA is mixedwith 2 μg of 25 kDa linear polyethylenimine as previously described(Durocher, Perret & Kamen, 2002) For every ml of CHO cells to betransfected, 1 μg of plasmid DNA is mixed with 8 μg of 25 kDa linearpolyethylenimine or with 6 μg of 40 kDa linear and deacetylated PEI(PEIMAX™ from Polysciences. Inc, catalog #24765-2).

Secreted alkaline phosphatase activity determination: SEAP activity ismeasured as previously described using the colorimetric substrateparanitrophenyl phosphate (Durocher et al, 2000).

Single Vector EBV oriP-EBNA1 Stable Expression System:

Mammalian cells transfected with an expression system in which a singlevector contains an Epstein-Barr virus nuclear antigen-1 (EBNA1)nucleotide sequence encoding a truncated EBNA1 protein, an Epstein-BarrVirus (EBV) oriP nucleotide sequence and a gene of interest unexpectedlyprovide enhanced stable expression of the gene of interest.

Truncated EBNA1 proteins include, for example, EBNA1c, EBNA1t andEBNA1s. These truncated forms are shown in FIG. 1. EBNA1t is a 417 aminoacid protein having DNA Linking Region 1 (LR1) containingTranscriptional Activation Domain (TAD) and DNA Linking Region 2 (LR2)without the Gly-Ala repeats of the 641 amino acid full length protein.EBNA1c is a 306 amino acid protein in which LR2 is present but LR1 isabsent, and EBNA1s is a 337 amino acid protein in which LR1 is presentbut LR2 is absent. EBNA1 nucleotide sequences encoding EBNA1c arepreferred in the vector. The amino acid sequences for the full lengthEBNA1 protein as well as the truncated forms EBNA1t, EBNA1c and EBNA1sare shown in SEQ ID NO: 1-4, respectively. The corresponding nucleotidesequences of the nucleic acid molecules encoding full length EBNA1 andthe truncated forms are shown in SEQ ID NO: 5-8, respectively.

All or any functional part of the complete nucleotide sequence may beused in the vector. The EBV oriP complete nucleotide sequence (pTT3vector) is shown in SEQ ID NO: 9, and a functional EBV oriP truncatednucleotide sequence (pTT5 vector) is shown in SEQ ID NO: 10. The oriPsequence comprises the Family of Repeats (FR) component and the dyadsymmetry (DS) component. The oriP sequence, particularly the FRcomponent, contributes to increased expression and stability ofexpression of the integrated truncated EBNA1 gene.

The gene of interest may be any gene that encodes a protein product ofinterest. Expression of the gene of interest in the transfected cellspermits stable, large-scale production of the protein product forindustrial purposes. Some particular genes of interest include, forexample, genes that encode monoclonal antibodies, erythropoietins,interferons, vascular endothelial growth factors, stem cell growthfactors, growth hormones, insulin-like growth factor binding proteins,etc.

The single vector also preferably comprises a selection gene to permitselection of the transfected cells for the propagation of stable celllines. Any suitable selection gene may be used. One example of a classof such genes are genes that confer antibiotic resistance on the cellwhen the vector is transfected into the cell. Some examples within thisclass include genes that confer resistance to puromycin, blasticidin,geneticin, zeocin or hygromycin. The blasticidin resistance cassette asfound in the pYD7 vector is particularly preferred. After transfectionof a cell with the vector, the cell may be permitted to replicate.Clones possessing the expression system may be selected on the basis ofthe selection gene, for example, by treating the cells with anantibiotic and culturing the cells that survive. In this way, a cellline stably expressing the gene of interest may be created.

Any or all of the nucleotide sequences and/or genes in the integratedvector may be under the control of a promoter also incorporated in thevector. Generally, each gene has its own promoter. Thus, there ispreferably a promoter for the EBNA1, a promoter for the gene of interestand a promoter for the selection gene. Strong or weak promoters may beused. Some promoters include, for example, the cytomegalovirus (CMV)promoter, Elongation Factor 1 alpha-HTLV (EF1α-HTLV) hybrid promoter,and Rous sarcoma virus (RSV) promoter. Also, any or all genes may have apolyadenylation signal. Alternatively, two genes, separated by anInternal Ribosome Entry Site (IRES), can be expressed by using only onepromoter and one polyadenylation signal.

Genetic maps of two embodiments (pTT31-SSH and pTT34-SSH) of the singlevector are shown in FIG. 3A. pTT31-SSH is a 8014 bp vector in which theEBNA1c gene is under the control of a strong promoter (EF1α-HTLV) andthe gene of interest (secreted alkaline phosphatase (SEAP) gene) isunder the control of a strong promoter (CMV). pTT34-SSH is a 8040 bpvector in which the EBNA1c gene is under the control of a weakerpromoter (RSV) and the gene of interest (secreted alkaline phosphatase(SEAP) gene) is under the control of a strong promoter (CMV).

FIG. 3B depicts a graph of SEAP activity in CHO cells transfected withpTT vectors (oriP-containing vectors) encoding EBNA1c in cis under aweak (RSV) or strong (EF1α-HTLV) promoter. Referring to FIG. 3B, CHOcells were transfected with increasing amounts of the SEAPgene-containing plasmids pTT-SSH (with oriP but without EBNA1c),pTT31-SSH (with oriP and with EBNA1c under the control of the strongEF1α-HTLV promoter) and pTT34-SSH (with oriP and with EBNA1c under thecontrol of the weak RSV promoter). Both EBNA1c-containing pTT vectors(pTT31-SSH and pTT34-SSH) lead to an increase in SEAP activity in CHOcells over the non-EBNA1c-containing vector (pTT-SSH). Use of a strongpromoter to control EBNA1 expression optimizes levels of transactivatingactivity thereby optimizing expression of the gene of interest.

The single vector EBV oriP-EBNA1 expression system is useful indifferent types of mammalian cells, for example, Chinese Hamster Ovary(CHO) cells, human embryonic kidney 293 (HEK293) cells, Madin-DarbyCanine Kidney (MDCK) cells, Vero cells and PER.C6™ cells, especially CHOcells.

Two Vector EBV oriP-EBNA 1 Stable Expression System:

Mammalian cells transfected with an expression system comprising twoseparate vectors, a first vector containing an Epstein-Barr virusnuclear antigen-1 (EBNA1) nucleotide sequence encoding a truncated EBNA1protein and an Epstein-Barr Virus (EBV) oriP nucleotide sequence, and asecond vector comprising a gene of interest and an Epstein-Barr Virus(EBV) oriP nucleotide sequence unexpectedly provide enhanced stableexpression of the gene of interest. The use of oriP in both vectorscontributes to stability of expression and increased expression of thegene of interest. To facilitate production of stable cell lines thatstably express the gene of interest, both vectors contain selectiongenes. Selection genes are described above. For example, a stable CHOcell clone expressing EBNA1c driven from an integrated oriP vectorcontaining a blasticidin resistance cassette (pYD7) stably expressed thegene of interest for over 6 months in the absence of selection.

Stable mammalian cell lines can be produced either by simultaneouslytransfecting a cell with both vectors and then propagating the cell, orby transfecting a cell with one of the vectors (either theEBNA1-containing or the gene of interest-containing vector) to produce astable clone and then transfecting a stable clone cell with the other ofthe vectors to produce a stable clone stably expressing the gene ofinterest.

Truncated EBNA1 proteins and corresponding genes, as well as the oriPand genes of interest are described above. As described previously,genes may be under the control of promoters. The two vector EBVoriP-EBNA1 stable expression system is also useful in different types ofmammalian cells, for example, Chinese Hamster Ovary (CHO) cells, humanembryonic kidney 293 (HEK293) cells, Madin-Darby Canine Kidney (MDCK)cells and PER.C6™ cells, especially CHO cells.

Referring to FIGS. 11 and 12, Chinese Hamster Ovary (CHO) cells stablyexpressing EBNA1c were produced by transfecting CHO cells with an EBNA1coriP-containing plasmid (pYD7 vector) using generally known methods witha linear 25 kDa polyethylenimine (PEI), and stable clones werepropagated. One CHO-EBNA1c clone (clone 3E7) so produced was transfectedwith secreted alkaline phosphatase (SEAP)-encoding and linearized pTT22(+oriP) or pTT30 (-oriP) vectors. The pTT22 and pTT30 vectors bothcontain a puromycin resistance cassette. The pTT22 vector contains oriP(i.e. the DS and FR elements are present) while the pTT30 vector doesnot contain oriP (i.e. the DS and FR elements are removed). CHO-EBNA1ccells transfected with the pTT22 and pTT30 vectors were transferred in96-well plates at a density of 100 cells/well. Puromycin was added 24hours post-transfection and selection was maintained for two weeks.

Referring to FIG. 11, after selection, SEAP activity (mOD/min at 410 nm)was measured in the supernatant at day 14 post-transfection. The resultsclearly demonstrate the transactivating action of EBNA1c on linearizedoriP-bearing expression plasmids integrated in CHO cells. Linearizationof the vector abolishes the replication potential of the oriP-EBNA1csystem thus eliminating the possibility that the increased SEAPexpression is due to plasmid replication. The average SEAP activity forthe 96 wells are 46.5 and 5.8 mOD/min for oriP-containing andnon-oriP-containing SEAP-encoding vectors, respectively.

Referring to FIG. 12, after selection, the best positive clones (7clones for pTT22-oriP vector and 6 clones for pTT30-non-oriP vector)were amplified and maintained in 6-well plates with or withoutpuromycin. Twenty days later, clones were seeded in a 6-well plate at0.25 million cells per ml and SEAP activity (OD/min at 410 nm) wasmeasured 5 days later. Also shown in FIG. 12 is the SEAP activity foundin the non-cloned (“pools” or “bulk”) transfected cells maintained inthe presence of puromycin for 34 days. SEAP activity in the oriP bulk is10 times higher than in the non-oriP bulk. These results clearlydemonstrate the transactivating action of EBNA1c on integratedoriP-bearing expression plasmids in CHO cells. For clones 4B10 and 4F4,the increased SEAP activity in the presence of puromycin suggests thatthese two clones are non-clonal and probably contaminated by a cellularpopulation expressing lower levels of SEAP. SEAP activity is expressedas increase in absorbance unit at 410 nm per min (OD/min).

Thus, the presence of oriP in the integrated expression plasmid confershigher expression levels of the gene of interest in EBNA1-expressingmammalian cells, particularly CHO cells, vs. non-oriP-containingplasmids.

Two Vector Transient Expression System in CHO Cells:

Chinese Hamster Ovary (CHO) cells transfected with an expression systemcomprising two separate vectors, a first vector containing anEpstein-Barr virus nuclear antigen-1 (EBNA1) nucleotide sequenceencoding a truncated EBNA1 protein and an Epstein-Barr Virus (EBV) oriPnucleotide sequence, and a second vector comprising a gene of interestand an Epstein-Barr Virus (EBV) oriP nucleotide sequence alsounexpectedly provide enhanced transient expression of the gene ofinterest in the CHO cells.

CHO cell lines can be produced either by simultaneously transfecting acell with both vectors and then propagating the cell, or by transfectinga cell with one of the vectors (either the EBNA1-containing or the geneof interest-containing vector) to produce a clone and then transfectinga clone cell with the other of the vectors to produce a clonetransiently expressing the gene of interest. Truncated EBNA1 proteinsand corresponding genes, as well as the oriP and genes of interest aredescribed above. As described previously, genes may be under the controlof promoters.

FIG. 2 illustrates transient expression of secreted alkaline phosphatase(SEAP) in CHO cells co-transfected with one vector containing oriP plusthe SEAP gene and another vector containing oriP plus truncatedEBNA1constructs (EBNA1t, EBNA1c and EBNA1s). In FIG. 2, CHO cells wereco-transfected with 50% of pTT-EBNA1constructs or 50% salmon testis DNA(stDNA) and (45% pTT-SEAP+5% pTT-GFP) plasmids. SEAP activity wascompared to activity in CHO cells transfected with 95% pTT-SEAP+5%pTT-GFP. SEAP activity (OD/min at 410 nm) was measured 5 dayspost-transfection. Transfection was accomplished using generally knownmethods with a linear 25 kDa polyethylenimine (PEI).

The results in FIG. 2 show an increase in transient SEAP activity of2-fold or higher in CHO cells co-transfected with oriP/EBNA1 andoriP/SEAP plasmids over CHO cells that are not co-transfected withoriP/EBNA1 plasmids. Further, while it has been previously shown thatthe “Transcriptional Activation Domain” (aa 65-89) in the LR1 domain ofEBNA1 is essential for transcriptional activation of integrated oriPvectors, FIG. 2 surprisingly shows that the truncated EBNA1c constructlacking the LR1 domain but containing the LR2 domain is capable ofincreasing gene expression from non-integrated oriP plasmids to the samelevel as EBNA1t (that contains both the LR1 and LR2 domains) or EBNA1s(that contains only the LR1 domain).

In FIG. 4, CHO cells were transfected with a linearized pTT-EBNA1cvector containing a blasticidin resistance cassette (pYD7 vector).Linearization of the vector was achieved following restriction enzymedigestion using Pvul enzyme. Following transfection, cells having stablyintegrated the pYD7 vector were selected by adding blasticidin to theculture medium. After a few days of blasticidin selection,blasticidin-resistant cells were seeded in 96-well plates withoutblasticidin selection. Emerging clones were tested for EBNA1cexpression. An EBNA1c-positive clone, 3E7 (FIG. 4A), was then selectedfor further testing. A master cell bank (MCB) and Working cell bank(WCB) were made at this point. The CHO-EBNA1c (clone 3E7) cells werecultured for over 130 days in the absence of blasticidin selection. Atvarious culture time points, an aliquot of the cells were taken forEBNA1c expression analysis by Western blot using an anti-EBNA1 antibody.FIG. 4B shows that the clone 3E7 is very stable over 130 days in culturewithout blasticidin selection pressure.

In FIG. 7, CHO cells containing integrated EBNA1-expressing plasmidswere produced by transfecting CHO cells with a pTT-EBNA1c vector, andthe clone propagated (clone 3E7). Resulting CHO-EBNA1c clone wastransfected with SEAP-encoding pTT plasmids with complete oriP(pTT-SMH), with DS-deleted oriP (pTTi-SMH), with FR-deleted oriP(pTTj-SMH) or with oriP-deleted (pTTl-SMH) pTT vectors. SEAP activity(OD/min at 410 nm) was measured in the supernatant at 5 dayspost-transfection. Transfections were accomplished using generally knownmethods with a linear 25 kDa polyethylenimine (PEI).

The results in FIG. 7 illustrate that increased expression intransiently transfected CHO-EBNA1cells is due to the transactivatingactivity of the oriP family of repeats (FR) element, and not to plasmidreplication. Removal of the dyad symmetry (DS) element (EBNA1-dependentorigin of replication) from the oriP does not inhibit expression whileremoving the FR element (responsible for EBNA1-dependent transcriptionalactivation) strongly reduces expression. The results also show that theDS element has a slight inhibitory effect on gene expression.

FIG. 8 compares the effect of the cytomegalovirus (CMV5) promoter andelongation factor 1 alpha-HTLV (EF1-αHTLV) promoter on transgeneexpression in CHO-EBNA1c cells (clone 3E7). CHO-EBNA1c-3E7 cells weretransfected with increasing amount SEAP-encoding oriP-containing (pTT)plasmids containing either the CMV5 or EF1-αHTLV promoter to control theSEAP gene (the overall content of DNA was kept constant by compensatingwith non-coding stDNA). SEAP activity (OD/min at 410 nm) was measured inthe supernatant at day 6 post-transfection. The results clearlydemonstrate that the CMV5 promoter is at least 5 times more potent thanthe EF1-αHTLV promoter at low plasmid doses (e.g. 5%). Further,CMV5-based plasmid needs 2-4 times less coding plasmid DNA for maximumexpression.

Codon-Optimized EBNA1 Constructs:

Codon-optimization of Epstein-Barr virus nuclear antigen-1 (EBNA1)nucleotide sequence strongly enhances expression of EBNA1 in mammaliancells, especially Chinese Hamster Ovary (CHO) and human embryonic kidney(HEK) cells. A codon-optimized EBNA1cDNA instead of non-codon-optimizedEBNA1cDNA may be used in any of the aspects of the present invention.Full length or truncated EBNA1cDNA may be codon-optimized.Advantageously, such codon-optimized EBNA1 nucleotide sequences permitthe use of weaker promoters to express EBNA1, thereby reducing thelikelihood of promoter competition between two strong promoters in asingle expression system. Codon-optimized EBNA1c nucleotide sequence(EBNA1c-CO, SEQ ID NO: 13) codes for a 308 amino acid protein (SEQ IDNO: 11). Codon-optimized EBNA1s nucleotide sequence (EBNA1s-CO, SEQ IDNO: 14) codes for a 337 amino acid protein (SEQ ID NO: 12).

Referring to FIG. 5, EBNA1constructs (EBNA1c and EBNA1s) werecodon-optimized (human codon usage—CO). pTT vectors containing EBNA1c,EBNA1s or their codon-optimized versions (EBNA1c-CO (SEQ ID NO: 13) andEBNA1s-CO (SEQ ID NO: 14)), and empty pTT vector (CTRL) were transfectedin separate HEK293 cells or CHO cells by generally known methods with alinear 25 kDa polyethylenimine (PEI). Three days post-transfection,cells were lyzed and cell extracts analyzed by Western blot using ananti-EBNA1 antibody. FIG. 5A is a Western blot for cell extracts fromHEK293 cells and FIG. 5B is a Western blot for cell extracts from CHOcells. In both cell lines it is evident that codon optimization enhancestransient expression of EBNA1 in the cells when compared to the control(CTRL) and the cells transfected with non-codon-optimized EBNA1.

For FIG. 5C, HEK293 cells were transfected with increasing amounts(1.25%, 5% and 20%) of pTT vectors containing EBNA1c, EBNA1c-CO, EBNA1sor EBNA1s-CO. Again it is evident from the Western blots in FIG. 5C thatcodon optimization enhances transient expression of EBNA1 in cells whencompared to cells transfected with non-codon-optimized EBNA1.

For FIG. 5D, CHO cells were co-transfected with pTT-SEAP (50%) plus 5%pTT-GFP vectors (Control) with or without 10% of pTT-EBNA1c,pTT-EBNA1cCO or pTT-VP16/EBNA1cCO (EBNA1cCO fused at its N-terminus toVP16—see below). Non-coding DNA (stDNA) was used as stuffer DNA tocomplete the amounts of DNA to 100%. SEAP activity measured 5 dayslater. This clearly demonstrates that, by improving its expression,codon optimization of EBNA1c provides an increased transactivatingactivity. A VP16-EBNA1cCO chimera also further increases transient geneexpression in CHO cells compared to EBNA1c and EBNA1cCO (see below).

EBNA1c-VP16 Fusion Protein:

A fusion protein comprising a truncated Epstein-Barr virus nuclearantigen-1c (EBNA1c) protein and a herpes simplex virus protein VP16provides significantly enhanced transactivating activity in mammaliancells, particularly Chinese Hamster Ovary (CHO) cells and humanembryonic kidney (HEK) cells.

A fusion protein is constructed by fusing VP16 to the N-terminus ofcodon-optimized EBNA1c. The VP16 cDNA encoding for the following proteinsequence was used: APPTDVSLGDELHLDGEDVAMAHADALDDFDLDMLGDGDSPGPGFTPHDSAPYGALDMADFEFEQMFTDALGIDEYGG (SEQ ID NO: 15). The VP16 cDNA sequence wascloned in-frame to the 5′ region of codon-optimized EBNA1c usinggenerally known methods.

The VP16-EBNA1cCO fusion protein in a pTT plasmid (10%) wasco-transfected in CHO cells with pTT-SEAP plasmid (50%) and pTT-GFPplasmid (5%) with a linear 40 kDa deacetylated polyethylenimine (seebelow). The CHO cells were transfected with 10% pTT/VP16-EBNA1cCO, 50%SEAP, 35% stDNA and 5% GFP. Non-coding DNA (stDNA) was used as stufferDNA to complete the amounts of DNA to 100%. Five days post-transfection,SEAP activity (OD/min) was measured and compared to activities in cellstransfected with stuffer DNA in place of pTT/EBNA1 vectors (CTRL), or apTT/EBNA1c vector or a pTT/EBNA1cCO vector in place of pTT/VP16-EBNA1c(FIG. 5D). The results in FIG. 5D clearly demonstrate that aVP16-EBNA1cCO chimera further increases transient gene expression in CHOcells compared to EBNA1c or EBNA1cCO.

Transfection with Fully Deacylated PEI:

Use of a 40 kDa fully deacetylated poly(ethylenimine) (LPEI-MAX) as atransfection reagent unexpectedly improves transfection efficiencyand/or productivity in Chinese Hamster Ovary (CHO) cells in comparisonto the use of the usual linear 25 kDa poly(ethylenimine) (LPEI). Such animprovement is not realized in human embryonic kidney (HEK) cells.

Referring to FIG. 6, HEK293-EBNA1t (clone 6E) and CHO-EBNA1c (clone 3E7)cells were used. For the upper panel of FIG. 6, HEK293-EBNA1t cells weretransfected with pTT-SEAP plasmids using LPEI-MAX at various DNA:PEIratios (R) and polyplexes amounts (%). SEAP activity (OD/min at 410 nm)was measured in the supernatant 6 days post transfection and compared tothe best condition found for LPEI (75% polyplexes; R=1:2). The resultsin the upper panel show that LPEI-Max is not better than LPEI in HEK293cells. For the lower panel, CHO-EBNA1cells were transfected withpTT-SEAP plasmids using LPEI-MAX at various DNA:PEI ratios. SEAPactivity (OD/min at 410 nm) was measured in the supernatant 6 days posttransfection and compared to the best condition found for LPEI (1:8).The results in the lower panel clearly demonstrate that LPEI-MAX issignificantly more potent than LPEI in CHO cells for transient geneexpression.

Co-Expression of FGF:

Co-expression of a fibroblast growth factor (FGF) increases heterologousgene expression in Chinese Hamster Ovary (CHO) cells.

Referring to FIG. 9, graphs are shown illustrating the effect of FGF2(bFGF) co-expression on transient gene expression in HEK293-EBNA1t(clone 6E) cells (upper panel) and CHO-EBNA1c (clone 3E7) cells (lowerpanel). The HEK293-EBNA1t and CHO-EBNA1c cells were transfected with 25%pTT-SEAP vector and increasing amounts (0%, 15%, 30%, 45%, 60% and 70%)of FGF2-encoding pTT plasmid (the overall content of DNA was keptconstant with non-coding stDNA). SEAP activity (OD/min at 410 nm) wasmeasured in the supernatant 7 days post-transfection. From the upperpanel it is evident that SEAP activity in HEK293-EBNA1t cells isdecreased by co-expression of FGF2. From the lower panel it is evidentthat SEAP activity in CHO-EBNA1c cells is increased by co-expression ofFGF2. This clearly demonstrates that the co-expression of FGF2 enhancestransgene expression in CHO cells but not in HEK293 cells. Increasedproductivity in CHO cells may be due to a FGF-induced rRNA synthesis.

PKB Potentiation of VPA:

Use of protein kinase B (PKB) to potentiate valproic acid (VPA)increases heterologous gene expression in mammalian cells, especiallyChinese Hamster Ovary (CHO) cells.

Valproic acid (VPA), a histone deacetylase inhibitor, enhances transientgene expression in cells. However, VPA also induces apoptosis therebykilling cells and reducing overall gains in productivity. It has nowbeen found that co-expressing PKB (also known as AKT) or aconstitutively active PKB mutant in the cells potentiates the action ofvalproic acid in gene expression by inhibiting apoptosis.

Referring to FIG. 10, the effect of PKB (AKT) and valproic acid (VPA) ontransient gene expression in CHO-EBNA1c (clone 3E7) cells isillustrated. The CHO-EBNA1c cells were transfected with a mixture ofSEAP-encoding oriP plasmids (pTT-SEAP) and stuffer DNA or pTT-AKTddvector (AKTdd is a dominant-positive mutant of AKT). In some cases, 0.25mM VPA was added 24 hours post-transfection. SEAP activity (ΔA410/min)was measured at days 5 to 8 post-transfection. FIG. 10 clearlydemonstrates that valproic acid increases transient gene expression inCHO cells and that transient co-expression of AKTdd greatly potentiatesthis effect.

REFERENCES

The contents of the entirety of each of which are incorporated by thisreference.

-   Mizuguchi H, Hosono T, Hayakawa T. Long-term replication of    Epstein-Barr virus-derived episomal vectors in the rodent cells.    FEBS Lett 2000 Apr. 28; 472(2-3):173-8.-   Durocher, Y, Perret, S, Thibaudeau, E, Gaumond, M H, Kamen, A,    Stocco, R, Abramovitz, M. A reporter gene assay for high-throughput    screening of G-protein-coupled receptors stably or transiently    expressed in HEK293 EBNA cells grown in suspension culture.    Analytical Biochemistry 2000 Sep. 10, 284 (2):316-26.-   Boussif O, Zanta M A, Behr J P. Optimized galenics improve in vitro    gene transfer with cationic molecules up to 1000-fold. Gene Ther    1996 December; 3(12):1074-80.-   Thomas M, Lu J J, Ge Q, Zhang C, Chen J, Klibanov A M. Full    deacylation of polyethylenimine dramatically boosts its gene    delivery efficiency and specificity to mouse lung. Proc Natl Acad    Sci USA 2005 Apr. 19; 102(16):5679-84.-   Kang M S, Hung S C, Kieff E. Epstein-Barr virus nuclear antigen 1    activates transcription from episomal but not integrated DNA and    does not alter lymphocyte growth. Proc Natl Acad Sci USA 2001 Dec.    18; 98(26):15233-8.-   Sears J, Kolman J, Wahl G M, Aiyar A. Metaphase chromosome tethering    is necessary for the DNA synthesis and maintenance of oriP plasmids    but is insufficient for transcription activation by Epstein-Barr    nuclear antigen 1. J Virol 2003 November; 77(21):11767-80.-   Kennedy G, Sugden B. EBNA1, a bifunctional transcriptional    activator. Mol Cell Biol 2003 October; 23(19):6901-8.-   Phiel C J, Zhang F, Huang E Y, Guenther M G, Lazar M A, Klein P S.    Histone deacetylase is a direct target of valproic acid, a potent    anticonvulsant, mood stabilizer, and teratogen. J Biol Chem 2001    Sep. 28; 276(39):36734-41.-   Chen J, Ghazawi F M, Bakkar W, Li Q. Valproic acid and butyrate    induce apoptosis in human cancer cells through inhibition of gene    expression of Akt/protein kinase B. Mol Cancer 2006; 5:71.-   Sheng Z, Liang Y, Lin C Y, Comai L, Chirico W J. Direct regulation    of rRNA transcription by fibroblast growth factor 2. Mol Cell Biol    2005 November; 25(21):9419-26.-   Kishida T, Asada H, Kubo K, Sato Y T, Shin-Ya M, Imanishi J,    Yoshikawa K, Mazda O. Pleiotrophic functions of Epstein-Barr virus    nuclear antigen-1 (EBNA1) and oriP differentially contribute to the    efficacy of transfection/expression of exogenous gene in mammalian    cells. Journal of Biotechnology 2007.-   Ettehadieh E, Wong-Madden S, Aldrich T, Lane K, Morris A E.    Over-expression of protein kinase Bα enhances recombinant protein    expression in transient systems. Cytotechnology 2002; 38:11-14.-   Krysan P J, Calos M P. Epstein-Barr virus-based vectors that    replicate in rodent cells. Gene 1993; 137-143.-   Tomiyasu K, Satoh E, Oda Y, Nishizaki K, Kondo M, Imanishi J,    Mazda O. Gene transfer in vivo and in vitro with Epstein-Barr    virus-based episomal vector results in markedly high transient    expression in rodent cells. Biochem Biophys Res Comm 1998; 253:    733-738.-   Goepfert U, Kopetzki E. Protein expression in rodent cells.    International Patent Publication WO 2007/048601 published 3 May    2007.-   Sunstrom N A, Kunaparaju R. Rodent expression system utilising    polyoma virus and Epstein-Barr virus sequences. International Patent    Publication WO 2005/024030 published 17 Mar. 2005.-   Durocher Y, Perret S, Pham P L, St-Laurent G, Kamen A. Enhanced    production of recombinant proteins by transient transfection of    suspension-growing mammalian cells. International Patent Publication    WO 2002/090533 published 14 Nov. 2002.-   Durocher Y. Expression vectors for enhanced transient gene    expression and mammalian cells expressing them. International Patent    Publication WO 2006/096989 published 21 Sep. 2006.

Other advantages that are inherent to the structure are obvious to oneskilled in the art. The embodiments are described herein illustrativelyand are not meant to limit the scope of the invention as claimed.Variations of the foregoing embodiments will be evident to a person ofordinary skill and are intended by the inventor to be encompassed by thefollowing claims.

1. An expression system for stable expression of a gene of interest, the expression system comprising one vector having an Epstein-Barr virus (EBV) nuclear antigen-1 (EBNA1) nucleotide sequence, a promoter and a polyadenylation signal for the EBNA1 nucleotide sequence, an EBV oriP nucleotide sequence, the gene of interest and a promoter and a polyadenylation signal for the gene of interest.
 2. The expression system of claim 1, wherein the EBNA1 nucleotide sequence encodes a truncated EBNA1 protein.
 3. The expression system of claim 1, wherein the EBNA1 nucleotide sequence is codon-optimized.
 4. A method of stably expressing a gene of interest in mammalian cells, the method comprising: transfecting a mammalian cell with an expression system as defined in claim 1; and, replicating the cell to provide mammalian cells that stably express the gene of interest.
 5. The method of claim 4, wherein the mammalian cells are selected from the group consisting of Chinese Hamster Ovary (CHO) cells, Human Embryonic Kidney (HEK) 293 cells, Madin-Darby Canine Kidney (MDCK) cells, Cero cells, and PER.C6™ cells.
 6. The method of claim 5, wherein the mammalian cells are CHO cells.
 7. An expression system for expression of a gene of interest, the expression system comprising: a first vector having an Epstein-Barr virus nuclear antigen-1 (EBNA1) nucleotide sequence, a promoter and a polyadenylation signal for the EBNA1 nucleotide sequence; and, a second vector having a gene of interest, a promoter and a polyadenylation signal for the gene of interest, and an EBV oriP nucleotide sequence.
 8. The expression system of claim 7, wherein the EBNA1 nucleotide sequence encodes a truncated EBNA1 protein.
 9. The expression system of claim 7, wherein the EBNA1 nucleotide sequence is codon-optimized.
 10. A method of expressing a gene of interest in mammalian cells, the method comprising: transfecting a mammalian cell with an expression system as defined in claim 6; and, replicating the cell to provide mammalian cells that express the gene of interest.
 11. The method of claim 10, wherein the mammalian cells are selected from the group consisting of Chinese Hamster Ovary (CHO) cells, Human Embryonic Kidney (HEK) 293 cells, Madin-Darby Canine Kidney (MDCK) cells, Cero cells, and PER.C6™ cells.
 12. The method of claim 10, wherein the mammalian cells are CHO cells.
 13. The method according to claim 9, wherein transfecting the cell with the first and second vectors is accomplished simultaneously, or the cell is transfected by one of the vectors first to produce a clone followed by transfection with the other vector to produce a clone that expresses the gene of interest.
 11. The method of claim 9, wherein the mammalian cell stably expresses gene of interest.
 12. The method of claim 11, wherein the mammalian cells are Chinese Hamster Ovary (CHO) cells.
 13. The method of claim 9, wherein the mammalian cell transiently expresses gene of interest.
 14. The method of claim 13, wherein the mammalian cells are Chinese Hamster Ovary (CHO) cells. 